Fig. 4: KIT is a critical downstream effector of TCOF1 in regulating CSC stemness.

a Heatmap of downregulated genes in TCOF1-depleted MDA-MB-468 mammospheres. Cutoff, Log2 FC < −1, GFOLD < −0.5. b RT-qPCR verified downregulation of KIT, FZD8 and NOS2 mRNAs in MDA-MB-468 mammospheres with TCOF1 knockout. Error bars, Mean ± SEM of three independent experiments. **p < 0.01; ***p < 0.001. c Immunoblotting showing expression levels of the indicated proteins in MDA-MB468 spheroids with or without TCOF1 knockout. d Expression levels of KIT in HCC1806 and HCC1143 spheroids with or without TCOF1 knockout. e Overexpression of HA-TCOF1 in MDA-MB-468 and HCC1806 spheroids. Whole-cell lysates were subjected to immunoblotting. f KIT mRNA levels in MDA-MB-468 spheroids overexpressing HA-TCOF1. Data represent mean ± SEM of three independent experiments, ***p < 0.001. g Mammosphere-formation assay of MDA-MB-468 cells upon KIT knockout. Error bars, mean ± SEM of three independent experiments. *p < 0.05. h, i MDA-MB-468 cells expressing HA-KIT or control vector were infected with tet-on TCOF1 or CTL gRNA. Cells were treated with dox (100 ng/ml) for 5 days and then subjected to mammosphere-formation assay. Error bars, mean ± SEM of three independent experiments. *p < 0.05. Whole-cell lysates were subjected to immunoblotting. j Ratio of secondary mammosphere number to primary mammosphere number. Data represents mean ± SEM of three independent experiments. *p < 0.05. Experiments in c–e were repeated twice independently with similar results. p values were calculated by two-sided Student’s t test in b, f, g, i, j.